Biochemical Tests for Reducing Sugars & Starch
- Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar solution to determine the concentration of reducing sugar present in the sample
- It is important that an excess of Benedict’s solution is used so that there is more than enough copper (II) sulfate present to react with any sugar present
- The intensity of any colour change seen relates to the concentration of reducing sugar present in the sample
- A positive test is indicated along a spectrum of colour from green (low concentration) to brick-red (high concentration of reducing sugar present)
- A semi-quantitative test can be carried out by setting up standard solutions with known concentrations of a reducing sugar (such as glucose)
- These solutions should be set up using a serial dilution of an existing stock solution
- Serial dilutions are created by taking a series of dilutions of a stock solution. The concentration decreases by the same quantity between each test tube
- They can either be ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm-3)
- Each solution is then treated in the same way: add the same volume of Benedict’s solution to each sample and heat in a water bath that has been boiled (ideally at the same temperature each time) for a set time (5 minutes or so) to allow colour changes to occur
- It is important to ensure that an excess of Benedict’s solution is used
- Any colour change observed for each solution of a known concentration in that time can be attributed to the concentration of reducing sugar present in that solution
- The same procedure is carried out on a sample with an unknown concentration of reducing sugar which is then compared to the standard solution colours to estimate the concentration of reducing sugar present
- To avoid issues with human interpretation of colour, a colorimeter could be used to measure the absorbance or transmission of light through the sugar solutions of known concentration to establish a range of values that an unknown sample can be compared against a calibration curve
Colorimeters
- A colorimeter is an instrument that beams a specific wavelength (colour) of light through a sample and measures how much of this light is absorbed (arbitrary units)
- They provide a quantitative measurement
- They contain different wavelengths or colour filters (depends on the model of colorimeter), so that a suitable colour can be shone through the sample and will not get absorbed. This colour will be the contrasting colour (eg. a red sample should have green light shone through)
- Remember that a sample will look red as that wavelength of light is being reflected but the other wavelengths will be absorbed
- Colorimeters must be calibrated before taking measurements
- This is completed by placing a blank into the colorimeter and taking a reference, it should read 0 (that is, no light is being absorbed)
- This step should be repeated periodically whilst taking measurements to ensure that the absorbance is still 0
- The results can then be used to plot a calibration or standard curve
- Absorbance against the known concentrations can be used
- Unknown concentrations can then be determined from this graph
A colorimeter is used to obtain quantitative data that can be plotted to create a calibration curve to be used to find unknown concentrations
